| Background:Epithelial ovarian cancer (EOC) is one of the malignant tumors that seriously threat women’s health. Because of insidious onset and rapid development, its mortality rate ranks the first among gynecological cancers. At present, cytoreductive surgery combined with platinum-based chemotherapy is effective in early stage of 70% of advanced stage patients, but the subsequent relapse and chemotherapy resistance often result in unimproved overall survival in ovarian cancer patients. The occurrence of chemotherapy resistance is an important reason for the poor prognosis. Therefore, a better understanding of the mechanism underlying the drug resistance in EOC is essential to improve the poor prognosis.The mechanisms of drug resistance to chemotherapy is very complex, and the possible mechanisms of cisplatin resistance include the pharmacokinetic factors (drug exposure and vascular tumor), micro-environmental factors (which are related to the impact on the cell cycle and apoptosis signaling) and cellular factors (such as the change in expression of proteins related to drug resistance, point mutations at drug target, enhanced cellular repair system and reduced apoptosis). Genetic and epigenetic variations in the key genes in these pathways can result in drug resistance in cancer cells as well. A large number of studies have also demonstrated that micro-RNAs are playing regulatory role in the expression of these key genes.Micro-RNA (miRNA) is a group of endogenous and non-coding nucleotides of about 22 nucleotides long and functions through complementary pairing with 3’-untranslated region (3’-UTR) of target gene to regulate the expression of target gene. MiR-100 as a potential tumor associated micro-RNA has been reported to be related to the occurrence,development and drug resistance of cancer. In our earlier study, we found that there is an association between the expression of miR-100 in EOC patients and clinical or pathological outcomes. We found that miR-100 is negatively correlated with the clinical stage, metastasis, relapse after chemotherapy and prognosis of ovarian cancer.Mammalian target of rapamycin (mTOR) and polo-like kinase 1 (PLK1) are members of highly conserved serine/threonine kinase family, which are widely present in eukaryotic cells, and are involved in cell proliferation, metabolism, metastasis and other biological processes. Previous studies have indicated that mTOR and PLK1 are the target molecules of miR-100. MiR-100 can repress the post-transcription of mTOR and PLK1 to control the growth, metastasis and chemotherapy sensitivity of tumor cells. Feng et al found that in lung adenocarcinoma, the down-regulation of miR-100 can activate target protein PLK1, thereby promoting taxol resistance. Zhu et al reported that in chondrosarcoma cell line miR-100 is targeted to the (3’-UTR) of mTOR to suppress its expression, which results in increased sensitivity of the cell line to cisplatin. There is also document on relationship between miR-100 and drug resistance in ovarian cancer to show that miR-100 can increase the sensitivity of ovarian clear carcinoma cell line to everolimus. However, it is not clear whether miR-100 is associated with cisplatin resistance in EOC.To explore the possible role of miR-100 in cisplatin resistance in EOC, we examined the expression of miR-100 in human ovarian cancer cell line SKOV3 and its cisplatin resistant cell line SKOV3/DDP. We up and down-regulated the expression of miR-100 in SKOV3/DDP to gain insights into the effect and mechanism of miR-100 regulating cisplatin resistance in epithelial ovarian cancer cells. And we build ovarian tumor model transplanted subcutaneously in nude mice to study the reversal effect of miR-100 on the drug resistance of epithelial ovarian cancer in nude mice and to explore the mechanism of its reversal action. These studies provide new insights into the mechanism of drug resistance in EOC, solution to overcome the drug resistance and help to identify new targets for the gene therapy of drug resistant ovarian cancer.Chapter 1 The experimental study on the reversal of cisplatin-resistance in ovarian epithelial carcinoma by miR-100 in vitroSection 1 The relationship between miR-100 and cisplatin resistance in epithelial ovarian cancer cellsObjective:To explore the possible role of miR-100 in cisplatin resistance in EOC, we examined the expression of miR-100 and cisplatin IC50 in human ovarian cancer cell line SKOV3 and its cisplatin resistant cell line SKOV3/DDP. To explore the regulatory effect of miR-100 on cisplatin resistance in epithelial ovarian cancer, we up and down-regulated miR-100 by transient transfection.Methods:1. Cell cultureSKOV3 and SKOV3/DDP cells were cultured in RPMI-1640 containing 10% fetal bovine serum at 37℃ and 5% CO2 in incubator with saturated humidity.2. Cell transient transfectionSKOV3/DDP cells were seeded in wells of 6-well plates at a density of 3.5×105 cells/well, cultured overnight. As cell confluence rate was 60-70%, the SKOV3/DDP cells were transfected with the mimics or inhibitor of miR-100 or negative control RNA (NC) or inhibitor negative control RNA (inhibitor NC) by lipofectamine 2000 according to the instructions. Then qRT-PCR and CCK8 experiments were carried out 24 hours after transient transfection.3. Expression of miR-100 of the cells in each group detected by qRT-PCR(1) Total RNA extraction:total RNA was extracted from cells using Trizol reagent; (2) The reverse transcription reaction:reverse transcription primer of miR-100 is a primer with stem loop structure, using U6 gene as reference. (3) real-time quantitative PCR:the reverse transcription products were used for PCR, setting up three holes.4. Cisplatin IC50 of the cells in each group detected by CCK8 experimentIn each group,cells were seeded in wells of 96-well plates at a density of 3×103 cells/well, cultured overnight and added with cisplatin at final concentration of 0 (control) to 64μg/mL. For each concentration, five wells were used. The cells were cultured for 48h and added with 10μg according to the manufacturer’s instructions. 4h later, the absorbance (A) at 450nm wavelength was measured using a plate reader to calculate the cell growth inhibition rate and half inhibitory concentration (IC50) for cisplatin.5. Statistical analyses were performed using SPSS version 20.0, and measurement data is expressed in mean differences± standard deviation (mean±SD). Comparison between two groups was analyzed by two-sample t test, and comparison of two in multiple groups was analyzed by One-Way ANOVA, P<0.05 was considered statistically significant.Results:1. Resistance of SKOV3/DDP cells to cisplatinWe first determined the IC50 to measure the resistance to cisplatin. For SKOV3/DDP and SKOV3 cells, IC50 was 8.29 and 3.72μg/mL respectively. Therefore, the resistance index of SKOV3/DDP cells to cisplatin was 2.23.2. Expression of miR-100 of the cells in each groupWe then profiled the expression of miR-100 in the two cell lines. It was found that the expression of miR-100 was 25 times less in SKOV3/DDP cells as compared with SKOV3 cells (P<0.001). However, after transfection with miR-100 mimices, SKOV3/DDP cells showed that the level of miR-100 was 38.29 times higher than that in the NC group (P<0.01), while transfection with miR-100 inhibitor resulted in significantly decreased miR-100 level by 97.7%in SKOV3/DDP cells (P<0.001)3. Cisplatin IC50 of the cells in each group after transient transfectionCell survival rate of SKOV3/DDP cells decreased with the increase of cisplatin concentration. After transfection with miR-100 mimices, The cisplatin IC50 of miR-100 mimices group was significantly lower than that in the NC group(P<0.001), while transfection with miR-100 inhibitor resulted in significantly increased cisplatin IC50 in SKOV3/DDP cells (P<0.001).Conclusion:1. SKOV3/DDP cells have drug resistance,and can stably passage. So the cells can be used for the follow-up experiment research.2. MiR-100 in the SKOV3/DDP cells was low expressed. MiR-100 is associated with cisplatin resistance in epithelial ovarian cancer, and it can increase the sensitivity of epithelial ovarian cancer cells to cisplatin.Section 2 The related mechanism of miR-100 regulating the sensitivity of epithelial ovarian cancer cells to cisplatinObjective:We construct epithelial ovarian cancer cell models with stably up and down-expression of miR-100 by lentivirus infection to gain insights into the mechanism of miR-100 regulating cisplatin resistance in epithelial ovarian cancer cells.Methods:1. Lentivirus infectionSKOV3/DDP cells were respectively infected with lentivirus (LV-miR-100, LV-anti-100, LV-NC).12h after infection, virus solution was removed and the cells were cultured in RPMI-1640 containing 10% fetal bovine serum for another 48h and examined for infection efficiency using fluorescence microscope, and further confirmed using qRT-PCR. The cells were cultured in the complete culture medium containing puromycin (2μg/mL) for two weeks to obtain SKOV3/DDP cells whose miR-100 was stably up-or down-regulated.2. Expression level of miR-100 in SKOV3/DDP cells after infection detected by qRT-PCRTotal RNA of SKOV3/DDP cells after lentivirus infection was respectively extracted, the expression level of miR-100 was detected by qRT-PCR. Largely amplify and frozen SKOV3/DDP cells with stably up- and down-regulated miR-100 expression and control cells for subsequent experiments.3. Growth curve assay(CCK8) was used to detect the impact of miR-100 on the growth state of SKOV3/DDP cellsSKOV3/DDP cells with stably up-and down-regulated miR-100 expression and control cells were seeded in wells of 96-well plates at a density of 2x103 cells/well respectively, five wells in each group, when cultured 12h、24h、36h、48h、72h, adding CCK8 10μL,37℃ cultured 2h, the absorbance (A) at 450nm wavelength was measured, blank control wells as zero, finally draw growth curves.4. Colony forming assay was used to detect the impact of miR-100 on the growth state of SKOV3/DDP cellsCells were inoculated to the wells of 6-well plates at a density of 1×102 cells/well and cultured for 14 days in RPMI-1640 medium with 10% fetal bovine serum, fixed in methanol, and stained with 0.1% crystal violet solution. Samples were photographed and counted for the number of visible colonies.5. Flow cytometry was used to detect the impact of miR-100 on early apoptosis of SKOV3/DDP cellsSKOV3/DDP cells with stably up- and down-regulated miR-100 expression and control cells were added with cisplatin (3μg/mL) and cultured for 48 h. The cells were then harvested and stained with FITC V and PI to be detected early apoptosis rate using flow cytometry.6. Flow cytometry was used to detect the impact of miR-100 on the cell cycle progression of SKOV3/DDP cellsSKOV3/DDP cells with stably up-and down-regulated miR-100 expression and control cells were collected and fixed in 70% ethanol on ice, washed with PBS, then stained with PI containing RNase A. Finally the cells were analyzed by flow cytometry.7. Western blot was used to detect the the regulation of miR-100 on mTOR and PLK1 proteinTotal protein was extracted, separated on SDS-PAGE gels, transferred to membranes and incubated with primary anti-bodies for 1h, and with secondary antibody for 1h. The membranes were developed using an enhanced chemiluminescence (ECL) kit and the gray values of the bands wee quantified using Quantity One software to calculate the relative expression level. The experiments were repeated 3 times.8. Statistical analyses were performed using SPSS version 20.0, and measurement data is expressed in mean differences± standard deviation (mean±SD). Comparison between two groups was analyzed by two-sample t test, and comparison of two in multiple groups was analyzed by One-Way ANOVA, P<0.05 was considered statistically significant.Results:1. Successfully produced cells models of SKOV3/DDP with stably up-and down-regulated miR-100 expression. The expression of GFP was about 90-95% under fluorescence microscope. The expression of miR-100 in SKOV3/DDP cells after lentivirus infection was detected by qRT-PCR. After infection with LV-miR-100, the level of miR-100 in the SKOV3/DDP cells was significantly higher than that in LV-NC-infected cells (P<0.05), while infection with LV-anti-100 resulted in significantly reduced miR-100 level in SKOV3/DDP cells(P<0.01).2. Growth curve assay(CCK8) was used to detect the impact of miR-100 on the growth state of SKOV3/DDP cellsAfter the infection with LV-miR-100, the proliferation of SKOV3/DDP cells was significantly reduced as revealed by the cell growth curve; on the other hand, infection with LV-anti-100 significantly increased the proliferation, as compared with LV-NC.3. Colony forming assay was used to detect the impact of miR-100 on the growth state of SKOV3/DDP cellsAfter the infection with LV-miR-100, the proliferation of SKOV3/DDP cells was significantly reduced as revealed by the colony numbers (P< 0.05); on the other hand, infection with LV-anti-100 significantly increased the proliferation, as compared with LV-NC (P< 0.05).4. Flow cytometry was used to detect the impact of miR-100 on early apoptosis of SKOV3/DDP cellsAfter 48h of cisplatin treatment at 3μg/mL, we examined the early apoptosis in SKOV3/DDP cells after infection. The results showed that the apoptosis was significantly higher in SKOV3/DDP cells infected with LV-miR-100 as compared with LV-NC-infected cells (P<0.01), while the apoptosis in SKOV3/DDP cells infected with LV-anti-100 was significantly reduced (P< 0.05).5. Flow cytometry was used to detect the impact of miR-100 on the cell cycle progression of SKOV3/DDP cellsThe proportions of cells at G1 or S phases in LV-miR-100-infected cells were significantly higher or lower than those in the LV-NC-infected cells (P<0.01), respectively; in contrast, the proportions of cells at G1 or S1 phases in the LV-anti-100-infected cells were significantly lower or higher than those in the LV-NC-infected cells (P< 0.05), respectively.6. Western blot was used to detect the the regulation of miR-100 on mTOR and PLK1 proteinWe examined the expression of mTOR and PLK1 in the cell lines before and after lentivirus infection using Western blot analysis. Compared with SKOV3 cells, mTOR and PLK1 levels were significantly higher in the resistant SKOV3/DDP cells (P< 0.05). After infection with LV-miR-100, the levels of the two proteins were significantly reduced as compared with the LV-NC-infected cells (P< 0.05), while infection with LV-anti-100 resulted in significantly higher expression of the two proteins (P<0.05).Conclusion:1. Successfully produced cells models of SKOV3/DDP with stably up-and down-regulated miR-100 expression and control cells by lentivirus infection.2. Up-regulation of miR-100 can inhibit cell proliferation, promote cell apoptosis and cell cycle arrest, and down-regulate mTOR and PLK1 expression in SKOV3/DDP cells. These may result in the increase of sensitivity to cisplatin in epithelial ovarian cancer cells by miR-100.Chapter 2 The experimental study on the reversal of cisplatin-resistance in ovarian epithelial carcinoma by miR-100 in vivoObjective:we build ovarian tumor model transplanted subcutaneously in nude mice to study the reversal effect of miR-100 on the cisplatin resistance of epithelial ovarian cancer and to explore the mechanism of its reversal action.Methods:1. Construction of the subcutaneous tumor model in nude miceCells in logarithmic growth were adjusted to a cell density of 5×107/mL and injected subcutaneously under the right or left shoulder without anesthesia of the mice at 200μL/mouse as approved by the Institutional Ethic Committee. The animals were monitored daily for the health conditions.2. Intraperitoneal injection of cisplatin in the treatment of subcutaneous transplanted tumor in nude mice and observation of the tumor growthWhen the tumors grew to 5mm in diameter cisplatin was injected at 4mg/kg every 3 days for a total of 7 times. The size of tumor was measured every 3 days. Three days after the end of treatment, the mice were sacrificed by cervical dislocation after being intraperitoneally injected with chloral hydrate to anaesthetize.3. The expression of mTOR and PLK1 protein of transplantation tumor in each group was detected by immunohistochemistryThe tumor tissues were collected, fixed in 10% formalin and used in immunohistochemistry assays for PLK1 and mTOR. Analyze the results of immunohistochemistry according to semiquantitative standard. Five fields were randomly selected and scored on the basis of cell staining and positive cell number.4. Statistical analyses were performed using SPSS version 20.0, and measurement data is expressed in mean differences± standard deviation (mean±SD). Comparison of two in multiple groups was analyzed by One-Way ANOVA, P<0.05 was considered statistically significant.Results:1. Successful construction of subcutaneous transplanted tumor model in nude mice. Pichugin disappeared about 3d after inoculation; subcutaneous tumors grew about 7d after inoculation; the rate of subcutaneous tumor formation was 100%.2. The growth of subcutaneous transplanted tumor in each group after cisplatin treatmentWhen the mice were grafted with SKOV3/DDP cells infected with LV-miR-100, the tumors grew slower than those grafted with LV-NC-infected SKOV3/DDP cells. On other hand, tumors grew faster in mice grafted with LV-anti-100-than with LV-NC-infected SKOV3/DDP cells.21 days after cisplatin treatment, the volumes of tumors derived from SKOV3/DDP cells that were stably infected with LV-miR-100 were significantly smaller than those from LV-NC-infected cells (P< 0.05). In contrast, the volumes of tumors derived from SKOV3/DDP cells that were stably infected with LV-anti-100 were significantly larger than those from with LV-NC-infected cells (P< 0.01).3. The expression of mTOR and PLK1 protein of transplantation tumor in each group was detected by immunohistochemistryImmunohistochemical analysis indicated that compared with tumors derived from LV-NC-infected cells, the tumors derived from LV-miR-100 or LV-anti-100 infected-cells had lower or higher mTOR and PLK1, respectively.Conclusion:MiR-100 can effectively reverse the cisplatin resistance of epithelial ovarian cancer in nude mice, and one of he mechanisms may be the down-regulation of mTOR and PLKl by miR-100. |
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